Space plasma physics: space experiments with particle accelerators.

Electron and plasma beams and neutral gas plumes were injected into the space environment by instruments on Spacelab 1, and various diagnostic measurements including television camera observations were performed. The results yield information on vehicle charging and neutralization, beam-plasma interactions, and ionization enhancement by neutral beam injection.

Sequential treatment with zoledronic acid followed by teriparatide or vice versa increases bone mineral density and bone strength in ovariectomized rats.

Bisphosphonates (BPs) and teriparatide (TPTD) are both effective treatments for osteoporosis, but BP treatment prior to daily TPTD treatment has been shown to impair the effect of TPTD in some clinical studies. In contrast, the loss of bone mineral density (BMD) that occurs after withdrawal of TPTD can be prevented by BP treatment. Although various studies have investigated the combination and/or sequential use of BP and TPTD, there have been no clinical studies investigating sequential treatment with zoledronic acid (ZOL) and TPTD (or vice versa). In this study, we evaluated the effects of sequential treatment with TPTD followed by ZOL, and ZOL followed by TPTD, using ovariectomized (OVX) rats. Two months after OVX, osteopenic rats were treated with ZOL, TPTD, or vehicle for a period of 4 months (first treatment period), and then the treatments were switched and administered for another 4 months (second treatment period). The group treated with ZOL followed by TPTD showed an immediate increase in BMD of the proximal tibia and greater BMD and bone strength of the lumbar vertebral body, femoral diaphysis, and proximal femur than the group treated with ZOL followed by vehicle. Serum osteocalcin, a marker of bone formation, increased rapidly after switching to TPTD from ZOL. The group treated with TPTD followed by ZOL did not lose BMD in the proximal tibia after TPTD was stopped, while the group treated with TPTD followed by vehicle did lose BMD. The BMD and bone strength of the lumbar vertebral body, femoral diaphysis, and proximal femur were greater in the group treated with TPTD followed by ZOL than in the group treated with TPTD followed by vehicle. The increase in serum osteocalcin and urinary CTX after withdrawal of TPTD was prevented by the switch from TPTD to ZOL. In conclusion, our results demonstrate that switching from ZOL to TPTD resulted in a non-attenuated anabolic response in the lumbar spine and femur of OVX rats. In addition, switching from TPTD to ZOL caused BMD to be maintained or further increased. If these results can be reproduced in a clinical setting, the sequential use of ZOL followed by TPTD or vice versa in the treatment of osteoporosis patients would contribute to increases in BMD that, hopefully, would translate into a corresponding decrease in the incidence of vertebral and non-vertebral fractures.

In vivo release and clearance of rat platelet phospholipase A2.

Intravenous injection of ADP into rats produced a rapid increase of plasma phospholipase A2 activity with a concomitant decrease in platelet count. Phospholipase A2 activity in plasma reached a peak within 1 min and thereafter declined sharply. This phospholipase A2 activity was reactive with a monoclonal antibody specific for rat platelet-derived phospholipase A2. These findings, together with the fact that ADP is a stimulant specific for platelets, suggest that the phospholipase A2 observed may be released into plasma from activated platelets in vivo. When platelet-derived phospholipase A2 was purified, labeled with 125I, and injected intravenously into rats, the radioactivity in the plasma decreased rapidly: the half-life of the enzyme in the blood stream was less than 30 s. Addition of either heparin or anti-phospholipase A2 monoclonal antibody directed against the domain of the enzyme responsible for binding to heparin retarded the clearance of the enzyme, suggesting that phospholipase A2 might be adsorbed onto heparan sulfate proteoglycans on the endothelial cell surface. Examination of the distribution of radioactivity in vivo revealed that most of the enzyme was rapidly taken up by the liver and degraded to acid-soluble materials.

Change in phospholipid composition of mouse bone marrow-derived mast cells during cultivation with fibroblasts.

A mouse bone marrow-derived mast cell (BMMC) clone, designated as MC-MKM, was established. MC-MKM cells released histamine and generated lipid chemical mediators such as prostaglandin D2 or platelet-activating factor upon stimulation with IgE and antigen. It is known that BMMCs acquired the characteristics of connective tissue mast cells (CTMCs) during coculture with fibroblasts. In the present study, we found that cellular phospholipid composition changed drastically when MC-MKM cells were cultured for 2 weeks in the presence of mouse 3T3 fibroblasts. In MC-MKM cells, phosphatidylcholine was the predominant phospholipid class, followed by phosphatidylethanolamine. During coculture with fibroblasts for 2 weeks, total phospholipid content in MC-MKM cells increased 2.5-fold. Among major phospholipid classes, a percentage of phosphatidylethanolamine increased most dramatically in accordance with a decrease in that of phosphatidylcholine, while no appreciable change in composition of other phospholipids was observed. The phospholipid composition of cocultured MC-MKM cells was closer to that of rat peritoneal CTMCs than that of the starting MC-MKM cells. The present findings that BMMCs converted their phenotype into CTMC-like cells by the interaction with fibroblasts in terms of phospholipid composition are not contradictory to the previous observations that BMMCs changed into CTMC-like cells.

Preferential hydrolysis of oxidized phospholipids by peritoneal fluid of rats treated with casein.

1-Palmitoyl-2-azelaoyl-PC, which is one of the possible cytotoxic products generated by the oxyhemoglobin-induced lipid peroxidation of 1-palmitoyl-2-linoleoyl-PC, was found to be efficiently hydrolyzed by the peritoneal fluid of rats treated with casein. The rate of hydrolysis of 1-palmitoyl-2-azelaoyl-PC was approx. 15-fold higher than that observed with 1-palmitoyl-2-linoleoyl-PC. When 1-palmitoyl-2-linoleoyl-PC pretreated with oxyhemoglobin was incubated with the peritoneal fluid, oxidized products of PC were hydrolyzed more efficiently than the intact 1-palmitoyl-2-linoleoyl-PC. When 1-[(1-)14C]palmitoyl-2-azelaoyl-PC was incubated with the peritoneal fluid, radiolabeled lysoPC was formed, whereas radiolabeled neutral lipids were not formed, indicating that the hydrolytic activity was of the 'phospholipase A2' type. We previously found and purified an extracellular phospholipase A2 (Chang, H.W. et al. (1987) J. Biochem. 102, 147-154) in the peritoneal fluid of rats injected intraperitoneally with casein. Hydrolysis of 1-palmitoyl-2-azelaoyl-PC by this purified phospholipase A2 was as low as that of 1-palmitoyl-2-linoleoyl-PC. These two phospholipase A2 activities showed different pH optima and Ca2+ requirements. The present phospholipase A2 activity, which preferentially hydrolyzes oxidized products of PC, may play an important role in detoxification or repair of damaged membrane in inflamed sites.

Cellular components that functionally interact with signaling phospholipase A(2)s.

Accumulating evidence has suggested that cytosolic phospholipase A(2) (cPLA(2)) and several secretory PLA(2) (sPLA(2)) isozymes are signaling PLA(2)s that are functionally coupled with downstream cyclooxygenase (COX) isozymes for prostaglandin (PG) biosynthesis. Arachidonic acid (AA) released by cPLA(2) and sPLA(2)s is supplied to both COX-1 and COX-2 in the immediate, and predominantly to COX-2 in the delayed, PG-biosynthetic responses. Vimentin, an intermediate filament component, acts as a functional perinuclear adapter for cPLA(2), in which the C2 domain of cPLA(2) associates with the head domain of vimentin in a Ca(2+)-sensitive manner. The heparin-binding signaling sPLA(2)-IIA, IID and V bind the glycosylphosphatidylinositol-anchored heparan sulfate proteoglycan glypican, which plays a role in sorting of these isozymes into caveolae and perinuclear compartments. Phospholipid scramblase, which facilitates transbilayer movement of anionic phospholipids, renders the cellular membranes more susceptible to signaling sPLA(2)s. There is functional cooperation between cPLA(2) and signaling sPLA(2)s in that prior activation of cPLA(2) is required for the signaling sPLA(2)s to act properly. cPLA(2)-derived AA is oxidized by 12/15-lipoxygenase, the products of which not only augment the induction of sPLA(2) expression, but also cause membrane perturbation, leading to increased cellular susceptibility to the signaling sPLA(2)s. sPLA(2)-X, a heparin-non-binding sPLA(2) isozyme, is capable of releasing AA from intact cells in the absence of cofactors. This property is attributed to its ability to avidly hydrolyze zwitterionic phosphatidylcholine, a major phospholipid in the outer plasma membrane. sPLA(2)-V can also utilize this route in several cell types. Taken together, the AA-releasing function of sPLA(2)s depends on the presence of regulatory cofactors and interfacial binding to membrane phospholipids, which differ according to cell type, stimuli, secretory processes, and subcellular distributions.

Immunochemical detection of 'platelet type' phospholipase A2 in the rat.

Polyclonal antibodies were raised against rat platelet phospholipase A2. One of them, designated as R377 was prepared by immunizing a rabbit with the intact enzyme. The other antibody, designated as R385, was prepared by immunizing with the enzyme treated with 2-mercaptoethanol. The antibody R377 bound to rat platelet phospholipase A2 almost exclusively, while the antibody R385 reacted not only with rat platelet phospholipase A2 but with the enzymes obtained from snake venom or mammalian pancreas. The antibody R377 bound to the non-reduced rat platelet phospholipase A2 bearing intact intramolecular disulfide bonds, but not to the reduced enzyme. In contrast, the antibody R385 reacted with both non-reduced and reduced enzymes. R377 may recognize the conformational structure of the enzyme. Both antibodies inhibited the enzyme activity. The antibody R377, but not R385, interfered with the interaction of the enzyme with heparin, which is one of the characteristic properties of rat platelet phospholipase A2. The antibody R377 reacted with phospholipases A2 of bone-marrow cells and of peritoneal exudated cells prepared from caseinate-treated rats, indicating that some myeloid cells other than platelets also contain 'platelet type' phospholipase A2. An immunochemical method for measurement of rat 'platelet type' phospholipase A2 was developed. The sensitivity of this method was 10 ng/ml of phospholipase A2 in the preparation. One of the advantages of the present immunochemical method is that the measurement was not affected by the presence of an endogenous inhibitor(s) of enzymatic activity.

Purification and characterization of rabbit platelet cytosolic phospholipase A2.

A phospholipase A2 was purified from rabbit platelet cytosolic fraction to near homogeneity by sequential column chromatographies on heparin-Sepharose, DEAE-Sephacel, butyl-Toyopearl, DEAE-5PW ion-exchange HPLC, and TSK gel G3000SW gel-filtration HPLC. The final preparation with an estimated specific activity of 8630 nmol/min per mg protein, showed a single band with a molecular mass of about 88 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver staining. The 88-kDa phospholipase A2 exhibited a fatty acid preference; it hydrolyzed phospholipid bearing an arachidonoyl residue at the sn-2 position more effectively than that with a linoleoyl residue. The catalytic activity of the purified enzyme with phosphatidylcholine or phosphatidylethanolamine increased sharply in the presence of between 10(-7) and 10(-6) M calcium ion, indicating that it could be regulated by less than micromolar concentration of calcium. These characteristics differ from those of platelet secretory 14-kDa phospholipase A2 reported previously. Therefore, this 88-kDa enzyme is a novel phospholipase A2 and may participate in the stimulus-dependent release of arachidonoyl residues in rabbit platelets.

Post-transcriptional regulation of LTC4 synthase activity by retinoic acid in rat basophilic leukemia cells.

Calcium ionophore-stimulated production of leukotriene (LT) C4 was enhanced by 16- to 26-h incubation with retinoic acid (RA) in rat basophilic leukemia-1 cells. Production of LTC4 by enzyme assay using cell lysates as the enzyme source and LTA4 as the substrate was also enhanced by RA-treatment. Production of LTB4 was not enhanced under these two experimental conditions, suggesting the preferential activation of LTC4 synthase activity. The RA-induced enhancement of LTC4 synthesis by the cells was suppressed by co-incubation with dexamethasone (DEX) or cyclosporine A (CSA). However, the expression of mRNA for LTC4 synthase was not affected by the exposure to RA, DEX or CSA. These results indicate that RA-induced enhancement of LTC4 production and its inhibition by DEX and CSA was determined by post-transcriptional regulation of LTC4 synthase.

Complete discrimination of docosahexaenoate from arachidonate by 85 kDa cytosolic phospholipase A2 during the hydrolysis of diacyl- and alkenylacylglycerophosphoethanolamine.

In our previous report (Shikano, M., Masuzawa, Y. and Yazawa, K. (1993) J. Immunol. 150, 3525-3533), we described that the enrichment of docosahexaenoic acid (DHA, 22:6(n - 3)) reduces both arachidonic acid (AA, 20:4(n - 6)) release and platelet-activating factor (PAF) synthesis in human eosinophilic leukemia cells, Eol-1. Since no DHA release was observed in response to Ca-ionophore stimulation, we presumed that the phospholipase A2 (PLA2) responsible for AA release and PAF synthesis can not hydrolyze the DHA moiety of phospholipids. In the present paper, we examined whether DHA-containing diacyl- and alkenylacylglycerophosphoethanolamine (DHA-diacylGPE and DHA-alkenylacyGPE) are susceptible to the action of AA-preferential 85 kDa cytosolic phospholipase A2 (cPLA2) from rabbit platelets in comparison with AA and eicosapentaenoic acid (EPA, 20:5(n - 3)) derivatives. When diacylGPE was used as a substrate, DHA release was almost negligible under the assay condition that allowed AA and EPA to be liberated at the rates of 4.3 mumol/min per mg protein and 2.5 mumol/min per mg protein, respectively. On the other hand, 14 kDa type II PLA2 hydrolyzed DHA-diacylGPE as well as AA-diacylGPE and EPA-diacylGPE. When DHA-diacylGPE and AA-diacylGPE were mixed at equimolar concentrations, DHA release by cPLA2 was not observed and AA release was reduced to 32% in the case without DHA-diacylGPE. This indicated that DHA-diacylGPE is a poor substrate but possesses the inhibitory activity for cPLA2. cPLA2 does not clearly discriminate between AA-alkenylacylGPE and AA-diacylGPE. As in the case using diacylGPE as a substrate, DHA-alkenylacylGPE was completely discriminated from AA-alkenylacylGPE by cPLA2. The roles of DHA and cPLA2 in the synthesis of lipid mediators will be discussed in relation to the new aspects of the substrate specificity of cPLA2 provided here.

Phospholipid composition of rat megakaryocytes and its rearrangement in platelets.

Rat platelets and their megakaryocyte precursors were examined for phospholipid composition. (1) The phospholipid composition of rat megakaryocytes, which were enriched and prepared from bone marrow cells, was almost identical to that of platelets. (2) The subclass composition of choline-containing glycerophospholipids (CGP) of rat megakaryocytes differed significantly from that of platelets: 1-alkenyl-2-acyl glycerophosphocholine (GPC) in megakaryocytes accounted for 29% of the total, whereas that in platelets was only 7%. (3) Rat platelets contained a larger amount of arachidonic acid than megakaryocytes, especially in ethanolamine-containing glycerophospholipids (EGP). (4) [32P]Phosphoric acid was significantly incorporated into megakaryocytes, whereas platelets showed little incorporation. On the other hand, the uptake of [3H]arachidonic acid into platelet phospholipids was about 15-times higher than that observed with megakaryocytes. (5) As reported previously for other blood cells, such as neutrophils and macrophages, the radioactivity of labeled arachidonic acid incorporated into CGP of platelets decreased, whereas that incorporated into EGP increased during a subsequent chase period. Hardly any such change was observed with megakaryocytes. These results suggest that the phospholipid composition of rat platelets is mainly determined at the time of thrombopoiesis, whereas the composition of molecular species is remodeled during circulation after thrombopoiesis.

The perturbed membrane of cells undergoing apoptosis is susceptible to type II secretory phospholipase A2 to liberate arachidonic acid.

Several lines of evidence have suggested that the plasma membranes of cells elicited by proinflammatory stimuli or microvesicles shed from activated cells are sensitive to extracellular type II secretory phospholipase A2 (sPLA2) that liberates fatty acids and lysophospholipids. Here we report that the membranes of cells undergoing apoptosis are highly susceptible to type II sPLA2. When neuronally differentiated rat pheochromocytoma PC12 cells deprived of nerve growth factor and serum, mouse mast cells deprived of hematopoietic cytokines or human monocytic U937 cells stimulated via Fas antigen (a receptor for the death factor Fas ligand), were exposed to type II sPLA2 at concentrations comparable to those detected at inflamed sites, the release of arachidonic acid was significantly accelerated in association with the process of programmed cell death. Arachidonic acid release by sPLA2 was dependent on the extracellular Ca2+ and was accompanied by preferential hydrolysis of phosphatidylethanolamine and phosphatidylserine in the membrane phospholipids. Association of sPLA2 with cell surface proteoglycan, which has been shown to be a prerequisite for endogenous sPLA2-dependent arachidonic acid release from the plasma membranes of live cells, was not essential for sPLA2-mediated hydrolysis of apoptotic cell membranes. Taking these results together, the apoptotic cell membrane is a potential target for extracellular type II sPLA2. The present findings may be relevant to events occurring at inflammatory or ischemic disease sites where apoptotic cells accumulate.

ESR study on synthetic glyceroglycolipid liposomal membranes.

We previously reported that glyceroglycolipid liposomes without cholesterol activated mouse peritoneal macrophages in vivo and in vitro, whereas glyceroglycolipid liposomes containing equimolar cholesterol did not. In order to characterize the properties of the glyceroglycolipid membranes, ESR spectroscopic studies were carried out with an acyl spin-labeled galactosyl ceramide (SL-GC) or a headgroup spin-labeled phospholipid (SL-6-DPPA) in 1,2-dipalmitoyl[beta-cellobiosyl-(1'---3)]glycerol (Cel-DAG) liposomal membranes. The ESR spectrum of the SL-GC in the Cel-DAG liposomes at 37 degrees C was a single broad line, indicating that the SL-GC molecules were excluded almost completely from Cel-DAG domains and formed clusters in the membranes. The spectrum of SL-6-DPPA in the Cel-DAG liposomes at 37 degrees C showed broad resonance lines with the central peak being the highest, while that at 60 degrees gave narrow lines with the low-field peak being the highest. This observation and rotational correlation time analysis showed that the molecular motions of spin-label moiety of the SL-6-DPPA were extremely restricted at 37 degrees C but not above Tc. These results suggest that below Tc the Cel-DAG molecules are packed tightly and restricted in motion in the membrane. Incorporation of cholesterol into the Cel-DAG liposomal membranes gave (1) the spectra of the SL-GC triplet, and (2) the spectra of the SL-6-DPPA narrow resonance with the low-field peak being the highest. These results suggest that cholesterol disturbs the rigid-packed structure of the Cel-DAG membrane and increases the molecular motions of the Cel-DAG. The DSC analysis of Cel-DAG with and without cholesterol agreed well to the results of the ESR technique. Thus we assume that peritoneal macrophages recognize the rigid-packed carbohydrate residues which are restricted in motion on the Cel-DAG membranes.

Detection and purification of two 14 kDa phospholipase A2 isoforms in rat kidney: their role in eicosanoid synthesis.

Phospholipase A2 (PLA2) activity in the soluble fraction of rat kidney yielded three peaks on DEAE cellulose column chromatography. From these three, we purified two PLA2 isoforms to near-homogeneity. Both had a molecular weight of approx. 14,000 on SDS-PAGE, and immunochemical and enzymological studies indicated that one is a 14 kDa type I PLA2 and the other a 14 kDa type II PLA2. RNA blot analysis confirmed that rat kidney contains both types of PLA2 and that administration of lipopolysaccharides and mercury chloride into rats increased type II PLA2 mRNA levels in kidney. When cultured rat mesangial cells were incubated with purified type I or type II PLA2 in combination with the calcium ionophore A23187 at suboptimal condition, augmentation of prostaglandin E2 production was observed. Type I and type II forms of PLA2 may play a role in arachidonate metabolism in rat kidney.

Lipoxygenase-catalyzed oxygenation of arachidonylethanolamide, a cannabinoid receptor agonist.

Various purified lipoxygenases were incubated with [14C]arachidonylethanolamide which is an endogenous ligand for cannabinoid receptors. When radioactive products were analyzed by thin-layer chromatography, porcine leukocyte 12-lipoxygenase and rabbit reticulocyte and soybean 15-lipoxygenases produced polar compounds at about the same reaction rates as that of oxygenation of free arachidonic acid. In contrast, the reaction of human platelet 12-lipoxygenase proceeded at a much lower rate, and porcine leukocyte 5-lipoxygenase was totally inactive. The result indicated that the lipoxygenases, which had been shown previously to be capable of oxygenating esterified polyunsaturated fatty acids, were also active with the arachidonylethanolamide. High-performance liquid chromatography, ultraviolet and mass spectrometry and nuclear magnetic resonance spectroscopy identified the major product by leukocyte 12-lipoxygenase as 12-hydroperoxy-5,8,10,14-eicosatetraenoylethanolamide and that by 15-lipoxygenases as 15-hydroperoxy-5,8,11,13-eicosatetraenoylethanolamide. The 15-hydroxy derivative inhibited electrically-evoked contraction of mouse vas deferens with an IC50 of 0.63 microM as well as arachidonylethanolamide (0.17 microM), but the 12-hydroxy derivative was much less effective.

Regulatory functions of phospholipase A2.

Phospholipase A2 (PLA2) plays crucial roles in diverse cellular responses, including phospholipid digestion and metabolism, host defense and signal transduction. PLA2 provides precursors for generation of eicosanoids, such as prostaglandins (PGa) and leukotrienes (LTs), when the cleaved fatty acid is arachidonic acid, platelet-activating factor (PAF) when the sn-1 position of the phosphatidylcholine contains an alkyl ether linkage and some bioactive lysophospholipids, such as lysophosphatidic acid (lysoPA). As overproduction of these lipid mediators causes inflammation and tissue disorders, it is extremely important to understand the mechanisms regulating the expression and functions of PLA2. Recent advances in molecular and cellular biology have enabled us to understand the molecular nature, possible function, and regulation of a variety of PLA2 isozymes. Mammalian tissues and cells generally contain more than one enzyme, each of which is regulated independently and exerts distinct functions. Here we classify mammalian PLA2s into there large groups, namely, secretory (sPLA2), cytosolic (cPLA2), and Ca(2+)-independent PLA2s, on the basis of their enzymatic properties and structures and focus on the general understanding of the possible regulatory functions of each PLA2 isozyme. In particular, the roles of type II sPLA2 and cPLA2 in lipid mediator generation are discussed.

Eicosanoid generation from antigen-primed mast cells by extracellular mammalian 14-kDa group II phospholipase A2.

The extracellular form of 14-kDa group II phospholipase A2 has been found to accumulate at various types of inflammatory sites. In the present paper, we have studied the possible role of the extracellular 14-kDa group II phospholipase A2 in the process of prostaglandin production in activated rat mast cells. When mast cells obtained from the peritoneal cavity of rats were sensitized with IgE, challenged with antigen and then exposed to extracellular 14-kDa group II phospholipase A2, appreciable release of prostaglandin D2 was observed. Generation of prostaglandin D2 was dependent on the concentration of the phospholipase A2 as well as that of the antigen, while no appreciable prostaglandin D2 generation was observed with cells in the absence of the antigen. No histamine release was observed under the same conditions. Phosphatidylcholine in mast cell membranes was appreciably hydrolyzed to liberate free arachidonic acid when mast cells were incubated with 14-kDa group II phospholipase A2 added exogenously in the presence of the antigen. Both the generation of prostaglandin D2 and the release of arachidonic acid were retarded by inhibitors specific to 14-kDa group II phospholipase A2. Thus, 14-kDa group II phospholipase A2 may function in the process of inflammation by acting on IgE-antigen-primed mast cells, which are not fully activated, to generate eicosanoids.

Functional crosstalk between phospholipase D(2) and signaling phospholipase A(2)/cyclooxygenase-2-mediated prostaglandin biosynthetic pathways.

We performed reconstitution analyses of functional interaction between phospholipase A(2) (PLA(2)) and phospholipase D (PLD) enzymes. Cotransfection of HEK293 cells with cytosolic (cPLA(2)) or type IIA secretory (sPLA(2)-IIA) PLA(2) and PLD(2), but not PLD(1), led to marked augmentation of stimulus-induced arachidonate release. Interleukin-1-stimulated arachidonate release was accompanied by prostaglandin E(2) production via cyclooxygenase-2, the expression of which was augmented by PLD(2). Conversely, activation of PLD(2), not PLD(1), was facilitated by cPLA(2) or sPLA(2)-IIA. Thus, our results revealed functional crosstalk between signaling PLA(2)s and PLD(2) in the regulation of various cellular responses in which these enzymes have been implicated.

Polyunsaturated fatty acids potentiate interleukin-1-stimulated arachidonic acid release by cells overexpressing type IIA secretory phospholipase A2.

By analyzing human embryonic kidney 293 cell transfectants stably overexpressing various types of phospholipase A2 (PLA2), we have shown that polyunsaturated fatty acids (PUFAs) preferentially activate type IIA secretory PLA2 (sPLA2-IIA)-mediated arachidonic acid (AA) release from interleukin-1 (IL-1)-stimulated cells. When 293 cells prelabeled with 13H]AA were incubated with exogenous PUFAs in the presence of IL-1 and serum, there was a significant increase in [3H]AA release (in the order AA > linoleic acid > oleic acid), which was augmented markedly by sPLA2-IIA and modestly by type IV cytosolic PLA2 (cPLA2), but only minimally by type VI Ca2(+)-independent PLA2, overexpression. Transfection of cPLA2 into sPLA2-IIA-expressing cells produced a synergistic increase in IL-1-dependent [3H]AA release and subsequent prostaglandin production. Our results support the proposal that prior production of AA by cPLA2 in cytokine-stimulated cells destabilizes the cellular membranes, thereby rendering them more susceptible to subsequent hydrolysis by sPLA2-IIA.

Phospholipase D is involved in cytosolic phospholipase A2-dependent selective release of arachidonic acid by fMLP-stimulated rat neutrophils.

When rat polymorphonuclear neutrophils (PMN) were treated with N-formyl-Met-Leu-Phe (fMLP), the release of arachidonic acid in preference to other fatty acids was observed. Levels of arachidonic acid reached a plateau within 5 min, and were accompanied by an approximately 4-fold increase in in vitro phospholipase (PL) A2 and PLD activities in PMN lysates. Treatment of PMN with ethanol (an inhibitor of PLD-mediated phosphatidic acid formation), propranolol (a phosphatidic acid phosphatase inhibitor), or 4-bromophenacylbromide (a PLA2 inhibitor), each suppressed fMLP-stimulated arachidonate release. Treatment with RHC-80267 (a diacylglycerol lipase inhibitor), however, had no such effect. The cytosolic PLA2 (cPLA2) inhibitor, arachidonoyl trifluoromethyl ketone, suppressed PLA2 activity in PMN homogenates and arachidonate release by fMLP-treated PMN. These results suggest that fMLP-elicited arachidonate release is mediated by cPLA2 but not diacylglycerol lipase, and that the activation of cPLA2 is downstream of the PLD-dependent signaling pathway.